KC-1293

Ba/F3-FIP1L1-PDGFRa-D842V-Cell-Line

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Background of Ba/F3-FIP1L1-PDGFRa-D842V-Cell-Line

Platelet-derived growth factor receptor A, abbreviated as PDGFRA or PDGFRα, is a cell-surface receptor tyrosine kinase, and activated by binding of its specific ligand, platelet-derived growth factors (PDGFs), play an important role in cell growth and differentiation. The overactivation of PDGFRA due to mutation and/or chromosome fusion are associated with hematological malignancy, and gastrointestinal stromal tumors (GISTs). The identification of PDGFRA as a driver gene has led to the development of anticancer therapeutics agents, including Avapritinib and Olaratumab. Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.

Specifications

Catalog Number	KC-1293
Cell Line Name	Ba/F3-FIP1L1-PDGFRa-D842V-Cell-Line
Description	Stable Ba/F3 clone expressing exogenous FIP1L1-PDGFRA fusion protein bearing D842V mutation in PDGFRA part.
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS+1µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Ba/F3 FIP1L1-PDGFRA-D842V cell line was generated using retrovirus vector expressing human FIP1L1-PDGFRA-D842V sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 1µg/mL Puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Montano-Almendras, C P, A Essaghir, H Schoemans, I Varis, L A Noel, A I Velghe, D Latinne, L Knoops, and J B Demoulin. 2012. ¡ùETV6-PDGFRB and FIP1L1-PDGFRA Stimulate Human Hematopoietic Progenitor Cell Proliferation and Differentiation Into Eosinophils: the Role of Nuclear Factor- B.¡ì Haematologica 97 (7): 1064Ã¿72.
Nielsen, B S, R R Malinda, F M Schmid, S F Pedersen, S T Christensen, and L B Pedersen. 2015. ¡ùPDGFR and Oncogenic Mutant PDGFR D842V Promote Disassembly of Primary Cilia Through a PLC - and AURKA-Dependent Mechanism.¡ì Journal of Cell Science 128 (19): 3543Ã¿49. doi:10.1242/jcs.173559.
Bahlawane, Christelle, Ren¹È Eulenfeld, Monique Y Wiesinger, Jiali Wang, Arnaud Muller, Andreas Girod, Petr V Nazarov, et al. 2015. ¡ùConstitutive Activation of Oncogenic PDGFRÏ«-Mutant Proteins Occurring in GIST Patients Induces Receptor Mislocalisation and Alters PDGFRÏ« Signalling Characteristics.¡ì Cell Communication and Signaling 13 (1): 49Ã¿21. doi:10.1186/s12964-015-0096-8.
Montano-Almendras, C P, A Essaghir, H Schoemans, I Varis, L A Noel, A I Velghe, D Latinne, L Knoops, and J B Demoulin. 2012. ¡ùETV6-PDGFRB and FIP1L1-PDGFRA Stimulate Human Hematopoietic Progenitor Cell Proliferation and Differentiation into Eosinophils: the Role of Nuclear Factor- B.¡ì Haematologica 97 (7): 1064Ã¿72. doi:10.3324/haematol.2011.047530.